Objective:
Elevated fat levels within tissues and reduced capacity/inefficiencies in long-chain fatty acid (LCFA) oxidative catabolism are highly correlated with muscle, liver, adipose, and whole-body insulin resistance and type 2 diabetes mellitus (T2DM). It is hypothesized that specific metabolites whose intra- or extra-cellular concentrations shift in response to changes in capacity or efficiency of mitochondrial LCFA combustion act as bioactive molecules that impact insulin signaling. We have leveraged metabolomics analysis platforms to discover new plasma metabolites that correlate with T2DM. One interesting outcome to date has been the identification of medium-chain fatty acylcarnitines (MCFA-carnitines, defined herein as C6-C14-carns and reflective of incomplete LCFA ß-oxidation) as entities increased in human T2DM plasma. Recent work has confirmed that (patho)physiologic concentrations of the natural isomer L-C14-carn triggers a suite of pro-inflammatory outcomes in multiple cell types. Since these inflammatory cascades have been implicated in attenuation of insulin signaling, it raises the possibility that intra- or extracellular accumulation of acylcarnitines could underlie or exacerbate the insulin resistance phenotype; if confirmed, this would represent a major paradigm shift. The molecular mechanisms by which acylcarnitines act to promote cytokine release, pro-inflammatory gene expression, and to activate inflammation cascades remain elusive, and this knowledge gap forms the basis of the current proposal. There is strong evidence that activation of pattern recognition receptors (PRRs)--at least TLR2, TLR4 and NOD2--can negatively impact insulin signaling. Thus, we propose to determine which specific PRR players are involved in acylcarnitine-mediated pro-inflammatory activity, and to determine for the first time if these factors induce insulin resistance.

Approach:
Specific Aim 1--Ascertain Whether the Pro-Inflammatory Effects of MCFA-Carnitine Result in Insulin Resistance in Skeletal Muscle and Adipocyte Preparations. Muscle and adipose tissues are important sites for glucose disposal and metabolism. Activators of PRRs and downstream JNK and NF'B activation have been associated with diminution of insulin signaling. Thus, in Aim 1 we will fully characterize the impact of exogenous acylcarnitines on insulin signaling cascades and glucose uptake in muscle preparations and in 3T3-L1 adipocytes. We hypothesize that the pro-inflammatory effects of L-C14-carnitine will have functional implications to reduce insulin action.
Specific Aim 2--Ascertain Whether the Pro-Inflammatory Effects of MCFA-Carnitine are Inhibited by PUFA via a GPR120-Dependent Pathway. Diets rich in '3-polyunsaturated fatty acids (PUFA) are known to dampen inflammation in vivo, and PUFAs attenuate the PRR-dependent pro-inflammatory effects of TLR activators in cell culture systems. Recently, it was reported that '3-PUFA (docosahexanoic acid, DHA) effects are largely via the GPCR GPR120 that inactivates nodes of the inflammatory cascade. Since acylcarnitines share some inflammation-associated systems with saturated FAs, in Aim 2 we will test the hypothesis that DHA will reduce acylcarnitine-induced inflammation. We further reason that such an activity, in part, will be driven by activation of the '3-PUFA receptor GPR120.
Specific Aim 3--Determine if MCFA-Carnitine Specifically Activates Intracellular Endosomal TLRs (TLR7, TLR8, TLR9). Our studies using murine and human cell systems and murine TLR gene constructs have successfully excluded several cell-surface PRRs as primary targets of L-C14-carnitine in terms of activation of pro-inflammatory pathways (e.g., TLR1-TLR6, see Preliminary Results). The effects of acylcarnitine in macrophage cells appear to involve MyD88, an adaptor protein shared by several TLRs. In Aim 3 we will test the hypothesis that L-C14-carnitine exerts its inflammatory effects through one or more of the MyD88-dependent endosomal PRRs TRL7, TLR8, or TLR9.